Microbial genomics for de-risking offshore

oil and gas exploration in Nova Scotia

Casey Hubert

Geomicrobiology Group

• marine oil spill bioremediation

• establishing environmental baselines

• seabed hydrocarbon seep microbiology

• deep biosphere microbial ecology

• extremophiles

• bacterial endospores

• oil reservoir microbiology

• food microbiology (coffee, sourdough)

Research Funding

Outline

1. original observations and interest in hydrocarbon seeps

2. using cold-adapted seabed microbes for prospecting

3. using dormant thermophilic bacterial spores for prospecting

Smeerenburgfjorden, Svalbard, 80° North

0

100

200

300

400

500

600

-5 0 5 10 15 20 25 30 35 40

Temperature (°C)

Svalbard marine sediment incubated from -2 to +40°C

CO2

organicsSO4-2

H2S

SRB

su

lph

ate

red

uction

ra

te (

nm

olcm

-3h

-1)

Bo Barker Jørgensen, unpublished

Thermophilic Bacteria in Arctic Sedimentssu

lfa

te r

ed

uctio

n r

ate

(%

of

op

tim

um

)

incubation temperature (°C)

Svalbard

~0°C

in-situ Hubert et al 2009 Science 325: 1541

SRB

sulfate-reducing bacteria

0 10 20 30 40 50 60 70 80

Thermophilic Bacteria in Arctic Sediments

incubation temperature (°C)

Svalbard

~0°C

in-situ

samples pre-pasteurized at 80°C

SRB

sulfate-reducing bacteria

0 10 20 30 40 50 60 70 80

0 10 20 30 40 50 60 70 80

su

lfa

te r

ed

uctio

n r

ate

(%

of

op

tim

um

) Hubert et al 2009 Science 325: 1541

Flemming Mønsted Christensen, M.Sc. thesis, 2009

Desulfotomaculum endospores

genus Desulfotomaculum

• sulfate reducers

• endospore formers

• thermophiles

CO2

simple

organicsSO4

-2

H2S

SRB

“sulfate reduction”

Flemming Mønsted Christensen, M.Sc. thesis, 2009

se

dim

en

t d

ep

th (

cm

)

se

dim

en

t d

ep

ositio

n (

ye

ar)

estimated abundance (thermophiles cm-3)

• 105 per gram of sediment

• annual influx 108 m-2

• where are they coming from?

Desulfotomaculum endospores

in Arctic marine sediment

Caloranaerobacter (2)

Caminicella

Desulfotomaculum

oil reservoir

mid-oceanridge

✓ warm

✓ anoxic

✓ fluid flow

Thermospores… where are they coming from?

rRNA genephylogeny:

5%

bottom water current

sedimentation

petroleum reservoirHubert & Judd 2010

Handbook of Hydrocarbon and Lipid Microbiology (Springer) Johnson et al. (2006) Geofluids 6: 251-271

Hydrocarbon seeps Hydrothermal circulation

• widespread in the ocean

• fluid seepage from depth

• depth influences in situ T

• thermospores have many

rRNA gene database hits to

oil fields

• worldwide network of ocean ridges

• lateral (axis) influence on in situ T

• thermospores have some rRNA

gene database hits to mid ocean

ridge systems

seabed fluid flow ~ an unappreciated microbial dispersal vector?

Caloranaerobacter (2)

Caminicella

Desulfotomaculum

✓ warm

✓ anoxic

✓ fluid flow

Thermospores… where do they come from?

rRNA genephylogeny:

5%

seabed fluid flow ~ an unappreciated microbial dispersal vector?

oil reservoir

mid-oceanridge

oil reservoir

bottom water current

sedimentation

Microbiology-based prospecting

for seabed hydrocarbon seeps

Microbiology-based prospecting

for seabed hydrocarbon seeps

oil reservoir

bottom water current

sedimentation

Microbiology-based prospecting

for seabed hydrocarbon seeps

oil reservoir

bottom water current

sedimentation

O2 conc.

oil reservoir

bottom water current

sedimentation

Hypothesis: thermophilic spores [ ] and cold-adapted

oil-degrading bacteria [ ] have different distributions at seeps

detectable range?

oil reservoir

bottom water current

sedimentation

Prospecting advantage using dormant bacterial spores

compared to active bacteria, or hydrocarbons…?

oil reservoir

sedimentation

Prospecting advantage using dormant bacterial spores

compared to active bacteria, or hydrocarbons…?

bottom water current

oil reservoir

bottom water current

sedimentation

Multiple approaches to exploration risk assessment

Outline

1. original observations and interest in hydrocarbon seeps

2. using cold-adapted seabed microbes for prospecting

3. using dormant thermophilic bacterial spores for prospecting

• Scotian Slope, 2500 - 3500 m

• Eastern Gulf of Mexico 1000 - 3000 m

Nova Scotia Department of Energy’s Play Fairway Analysis

• $15M investment by NS interdisciplinary geoscience mapping

• >$2B in exploration commitments (Shell, BP, Statoil)

• Front-end science to characterize the deep seabed reduced risk

Canadian Coast Guard

Ship: Hudson

summer 2015 & 2016

Scotian Slope – Piston Coring

>60 sites sampled

• 1750 to 3450 m water depth

• 0 to 10 mbsf sediment depth

Geochemical analyses (APT)

• Organic carbon content

• Gas wetness

• Isotopes (δ13C, δD)

• Sulfate depth profiles (UofC)

Microbial analysis

• >400 samples

• bacterial community

composition

Distinct sulfate profiles for Sites M, J, E

Oye Adebayo

Hydrocarbons = sulfate reduction 0

100

200

300

400

500

600

700

800

900

1000

0 20

Dep

th (

cm

)

Sulfate (mM)

Site M

Site J

Site E

Site N

Site H

SRB

sulfate-reducing bacteria

+

+

+

–

–

Geochem (APT)

Surface and Subsurface samples for DNA

0

100

200

300

400

500

600

700

800

900

1000

0 20

Dep

th (

cm

)

Sulfate (mM)

Surface: 0 cm & 20 cm

(trigger weight core and

piston core)

Subsurface: >20 cm

(only piston core)

subsurface (>20 cm) Atribacteria at 14 sites

A B C D E F I JH K L M N O0

10

20

30

40

50

60

70

80

Rel

ativ

e ab

unda

nce

(%)

Aerophobetes

Aminicenantes

Atribacteria

Atribacteria

Atribacteria

Atribacteria

Atribacteria

– – + + +– + –

– – – – + – – +– – – + – –

sulfate (U of C)

geochem (APT)

OTU_1

OTU_2

OTU_4984

OTU_58

OTU_9

7 dominant OTUs

Carmen Li

Deidra Stacey

G

7 dominant OTUs

OTU_2 0

5

10

15

20

25

30

35

40

Rel

ativ

e ab

unda

nce

(%)

Chloroflexi

Acidobacteria

Gammaproteobacteria

Alphaproteobacteria

Gammaproteobacteria

Aminicenantes

Atribacteria

A B C D E F I JH K L M N O

– – + + +– + –

– – – – + – – +– – – + – ––

sulfate (U of C)

geochem (APT)

Carmen Li

Deidra Stacey

subsurface (<20 cm) Atribacteria at 15 sites

0

5

10

15

20

25

30

35

40

Rel

ativ

e ab

unda

nce

(%)

Chloroflexi

Acidobacteria

Gammaproteobacteria

Alphaproteobacteria

Gammaproteobacteria

Aminicenantes

Atribacteria

subsurface (<20 cm) Atribacteria at 15 sites

A B C D E F I JH K L M N O

– – + + +– + –

– – – – + – – +– – – + – –

G

–

sulfate (U of C)

geochem (APT)

7 dominant OTUs

OTU_2

Carmen Li

Deidra Stacey

What is the functional role of uncultivated

Atribacteria? Metagenomics

O

N

OG

N

N

O

3 metagenomes

site 26 (O)

site 29 (OG)

site 44 (N)

• 58 draft genomes

from three samples

• 6 Atribacteria

preliminary evidence

reveals hydrocarbon

degradation genes in

5/6 Atribacteria

Xiyang Dong

Outline

1. original observations and interest in hydrocarbon seeps

2. using cold-adapted seabed microbes for prospecting

3. using dormant thermophilic bacterial spores for prospecting

seep-associated Atribacteria in

both Nova Scotia and Gulf of

Mexico sediments may be gas

or oil associated, and may be

capable of hydrocarbon

biodegradation

ongoing detailed analysis of

genomic and oil geochemistry

datasets

And! using genomics for exploration automatically

provides an environmental baseline

Deidra Stacey

optimizing conditions for baseline data collection

2 Nova Scotia cores × triplicate sediment aliquots

× 3 DNA extraction protocoss× 3 PCR primer pairs

Outline

1. original observations and interest in hydrocarbon seeps

2. using cold-adapted seabed microbes for prospecting

3. using dormant thermophilic bacterial spores for prospecting

Caloranaerobacter (2)

Caminicella

Desulfotomaculum

✓ warm

✓ anoxic

✓ fluid flow

Thermospores… where do they come from?

rRNA genephylogeny:

5%

Are spore-forming Firmicutes really prevalent in oil reservoirs?

Are spore-forming Firmicutes really prevalent in oil reservoirs?

Daniel Gittins

•  ~75 studies with amplicon libraries from oil reservoirs

•  only 12 from formation water (no water injection)

•  therefore truly indigenous communities analysed

•  Firmicutes indeed prevalent

•  next step: expand the study to include produced water samples as well as formation waters

111 surface sediments heated to 50°C

Anirban

Chakraborty

71 oil-positive and 40 oil-negative

Thermophilic Bacteria in cold sediments

incubation temperature (°C)

Svalbard

~0°C

in-situHubert et al 2009 Science 325: 1541

SRB

sulfate-reducing bacteria

0 10 20 30 40 50 60 70 80

(% o

f o

ptim

um

)

su

lfate

red

ucti

on

rate

incubation time (hours at 50oC)

50°C

(nm

olcm

-3h

-1) DNA sequencing to characterize

the microbial community after

several days at 50°C

1-30 thermospore

OTUs per sediment

115 thermospores detected @ 50°CT

herm

osp

ore

OT

Us

site occupancy

top 12 oil-associated

thermospores in EGoM

• ≥80% detection in

oil-positive locations

oil-positive

oil-negative

top 12 oil-associated

thermospores in EGoM

• ≥80% detection in

oil-positive locations

oil-positive

oil-negative

top 12 oil-associated

thermospores in EGoM

• ≥80% detection in

oil-positive locations

oil-negative

Outline

1. original observations and interest in hydrocarbon seeps

2. using cold-adapted seabed microbes for prospecting

3. using dormant thermophilic bacterial spores for prospecting

Conclusion: mutually

exclusive strategies for

seep detection are showing

promising results

oil reservoir

bottom water current

sedimentation

other applications?

thermospores as messengers from the reservoir?